Inhibition of CYP2D6 with low dose (5 mg) paroxetine in patients with high 10-hydroxynortriptyline serum levels-A prospective pharmacokinetic study.

The antidepressant nortriptyline is metabolized by cytochrome P450 2D6 (CYP2D6) to the less active and more cardiotoxic drug metabolite, 10-hydroxynortriptyline. High serum levels of this metabolite (>200 µg/L) may lead to withdrawal of nortriptyline therapy. Adding CYP2D6 inhibitors reduce the metabolic activity of CYP2D6 (phenoconversion) and so decrease the forming of hydroxynortriptyline. In this study, 5 mg paroxetine is administered to patients with high hydroxynortriptyline concentrations (>200 µg/L). The shift in number of patients to therapeutic nortriptyline (50-150 µg/L) and safe hydroxynortriptyline (<200 µg/L) concentrations, and the degree of phenoconversion, expressed as the change in ratio nortriptyline/hydroxynortriptyline concentrations before and after paroxetine addition, are prospectively observed and described. After paroxetine addition, 12 patients (80%) had therapeutic nortriptyline and safe hydroxynortriptyline concentrations. Hydroxynortriptyline concentrations decreased in all patients. The average nortriptyline/hydroxynortriptyline concentrations ratio increased from 0.32 to 0.59. This study shows that 5 mg paroxetine addition is able to lower high hydroxynortriptyline serum levels to safe ranges.

Practical liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of amitriptyline, nortriptyline and their hydroxy metabolites in human serum.

Amitriptyline (AMI) has been in use for decades in treating depression and more recently for the management of neuropathic pain. A highly sensitive and specific LC-tandem mass spectrometry method was developed for simultaneous determination of AMI, its active metabolite nortriptyline (NOR) and their hydroxy-metabolites in human serum, using deuterated AMI and NOR as internal standards. The isobaric E-10-hydroxyamitriptyline (E-OH AMI), Z-10-hydroxyamitriptyline (Z-OH AMI), E-10-hydroxynortriptyline (E-OH NOR) and Z-10-hydroxynortriptyline (Z-OH NOR), together with their parent compounds, were separated on an ACE C18 column using a simple protein precipitation method, followed by dilution and analysis using positive electrospray ionisation with multiple reaction monitoring. The total run time was 6 min with elution of E-OH AMI, E-OH NOR, Z-OH AMI, Z-OH NOR, AMI (+ deuterated AMI) and NOR (+ deuterated NOR) at 1.21, 1.28, 1.66, 1.71, 2.50 and 2.59 min, respectively. The method was validated in human serum with a lower limit of quantitation of 0.5 ng/mL for all analytes. A linear response function was established for the range of concentrations 0.5-400 ng/mL (r2 > .999). The practical assay was applied on samples from patients on AMI, genotyped for CYP2C19 and CYP2D6, to understand the influence of metaboliser status and concomitant medication on therapeutic drug monitoring.

Genetic differences in cytochrome P450 enzymes and antidepressant treatment response.

AIMS: Antidepressant response varies between patients, possibly due to differences in the rate cytochrome P450 enzymes metabolise antidepressants into inactive compounds. Drug metabolism rates are influenced by common variants in the genes encoding these enzymes. However, it remains unclear whether treatment outcomes can be predicted by either CYP450 genotype or antidepressant serum concentration. METHODS: In GENDEP (a pharmacogenetic study of depressed individuals treated with either escitalopram or nortriptyline), serum concentrations of antidepressants and their primary metabolite were measured after eight weeks treatment and variants in CYP2D6 and CYP2C19 were genotyped. RESULTS: Amongst patients taking escitalopram (n=223), the genotype CYP2C19 was significantly associated with escitalopram serum concentrations and desmethylescitalopram:escitalopram ratio. For those taking nortriptyline (n=161), the CYP2D6 genotype was significantly associated with nortriptyline and 10-hydroxynortriptyline serum concentrations and 10-hydroxynortriptyline:nortrip-tyline ratio. CYP450 genotypes conferring greater enzyme activity were linked to lower drug serum concentrations and higher metabolite:drug ratios. Nonetheless, no significant association was found between either CYP450 genotype or antidepressant serum concentration and treatment response. CONCLUSIONS: While there is a significant relationship between the CYP450 genotype and serum concentrations of escitalopram and nortriptyline, the genotypes are not predictive of differences in treatment response for either drug. Furthermore, differences in antidepressant serum concentrations are not associated with variability in treatment response.

Fast and sensitive LC-MS/MS assay for quantification of nortriptyline and its active metabolites E- and Z-10-hydroxynortriptyline in human plasma.

BACKGROUND: A fast and sensitive validated assay for nortriptyline, E-10-hydroxynortriptyline and Z-10-hydroxynortriptyline in plasma following a single oral dose of nortriptyline 25 mg was needed to support a clinical study. RESULTS: Plasma samples were prepared by protein precipitation, separated on a C18 column with a mobile phase consisting of 0.1% formic acid in an acetonitrile gradient over 6 min and detected by ESI in the positive mode and MS/MS. Mean recoveries of at least 90% were achieved. The LLOQ was 0.2 ng/ml for nortriptyline and 0.5 ng/ml for the metabolites. The standard curve was linear within LLOQ to 40 ng/ml (r(2) ≥ 0.997), precision was under 7.1% coefficient of variance (<16% at LLOQ) and accuracy was 92-114%. CONCLUSION: A fast and sensitive assay for nortriptyline, E- and Z-10-hydroxynortriptyline in plasma was developed and validated. It has been applied successfully to a clinical study.

Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of nortriptyline and 10-hydroxynortriptyline in human plasma: application to a human pharmacokinetic study.

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of nortriptyline (NTP) and 10-hydroxynortriptyline (OH-NTP) in human plasma (250 µL) using carbamazepine as an internal standard (IS). LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. A simple liquid-liquid extraction process was used to extract NTP, OH-NTP and IS from human plasma. The total run time was 2.5 min and the elution of NTP, OH-NTP and IS occurred at 1.44, 1.28 and 1.39 min, respectively; this was achieved with a mobile phase consisting of 20 mm ammonium acetate : acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPURITY C(18) column. The developed method was validated in human plasma with a lower limit of quantitation of 1.09 ng/mL for both NTP and OH-NTP. A linear response function was established for the range of concentrations 1.09-30.0 ng/mL (r > 0.998) for both NTP and OH-NTP. The intra- and inter-day precision values for NTP and OH-NTP met the acceptance as per FDA guidelines. NTP and OH-NTP were stable in a battery of stability studies, i.e. bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in humans.

Disputed case of homicide by smothering due to severe amitriptyline intoxication of the victim.

We report a fatal case of a female for whom the forensic autopsy revealed injuries to the external respiratory orifices indicating smothering. Subsequent postmortem toxicological analysis confirmed heavy amitriptyline acute intoxication. The victim had serious psychological problems, was under long-term treatment with antidepressants and was a systematic alcohol abuser. Forensic autopsy determined damage to the external airways, along with multiple formal petechial hemorrhages (Tardieu) in various parts of the body. The presence of amitriptyline, nortriptyline and 10-hydroxynortriptyline was confirmed by GC-MS and quantified by HPLC in blood (7.0 microg/ml amitriptyline and 7.4 microg/ml nortriptyline). The cause of death was disputed between severe intoxication (poisoning or suicide attempt) and smothering due to controversial evidence.

[Influence of ORM1 polymorphism on serum concentration of free nortriptyline].

To study the effect of alpha1-acid glycoprotein 1 (ORM1) polymorphism on the concentration of free nortriptyline in serum, genotyping analysis was employed in ORM1 by sequencing. Eighteen unrelated male adults were chosen and given a single dose of 25 mg nortriptyline orally, then the blood samples were taken at 0, 1, 2, 3, 4, 6, 8, 12, 24, 32, 48, 72, 96 and 168 hours after drug administration. Nortriptyline and 10-OH-nortriptyline in serum and ultrafiltrate were detected for the total and free concentration by using HPLC-MS/MS. Pharmacokinetic parameters were compared among different ORM1 genotypes. No significant differences were shown in the pharmacokinetic parameters of total nortriptyline and 10-OH-nortriptyline. The mean AUC(0-infinity) of free nortritpyline in ORM1 * F/ * F1 subjects was significantly higher than that in ORM1 * F1/ * S and ORM1 * S/ * S subjects [(119.1 +/- 74.4) ng x mL(-1) x h vs (51.4 +/- 23.2) ng x mL(-1) x h and (42.4 +/- 11.6) ng x mL(-1) x h]. The percentage of protein binding in subjects with ORM1 * F1/ * F1 genotype at 2, 3, 4, 6, 8 and 12 h after administration was slightly lower than in those with ORM1 * F1/ * S and ORM1 * S/ * S genotypes while the distinct difference was shown at 4 h (P < 0.05). Different ORM1 genotypes might affect the protein binding percentage and the concentration of serum free nortriptyline. The ability binding to the drug was higher in subjects with ORM1 * S/ * S genotype than in those with other two genotypes, so as to cause the lower concentration of free nortriptyline.

Placental passage of tricyclic antidepressants.

BACKGROUND: The use of antidepressants during pregnancy continues to garner considerable attention, though there are limited investigations that have sought to quantify fetal exposure. METHODS: Maternal and umbilical cord sera were collected at delivery from ten women taking nortriptyline and seven taking clomipramine. Placental passage was calculated as the ratio of umbilical cord to maternal serum concentration. Obstetrical outcome data were gathered from subjects at delivery. RESULTS: The placental passage ratio of nortriptyline and its active metabolite, cis-10-hydroxynortriptyline, were .68 +/- .40, 1.40 +/- 2.40, respectively. Clomipramine and desmethylclomipramine ratios were .60 +/- .50, .80 +/- .60. Obstetrical complications, such as pre-term delivery and pregnancy induced hypertension, were increased compared to the national average. CONCLUSIONS: The in vivo ratios of umbilical cord to maternal serum drug concentrations demonstrate considerable fetal exposure and differ greatly from previous results utilizing ex vivo perfusion.

Influence of P-glycoprotein inhibition on the distribution of the tricyclic antidepressant nortriptyline over the blood-brain barrier.

The distribution of the antidepressant drug nortriptyline (NT) and its main metabolite E-10-hydroxy-nortriptyline (E-10-OH-NT) across the blood-brain barrier was considered in relation to inhibition of the multidrug transporter P-glycoprotein (P-gp). Rats received NT in doses of 25 mg/kg orally, 10 mg/kg i.p. or 25 mg/kg i.p. Half the rats were treated with the P-glycoprotein inhibitor cyclosporine A (CsA) (200 mg/kg) 2 h prior to NT administration, and the other half served as a control group. NT and the metabolite were extracted from brain and serum by liquid-liquid extraction and analysed by HPLC with UV-detection. The brain to serum ratio of NT was increased in the CsA treated groups (22.3-26.8) compared with the control groups (16.5-22.7), the difference being statistically significant in two of the three experiments (p<0.05). Increased brain-serum ratios were also found for E-10-OH-NT, but the differences were not statistically significant. These results suggest that inhibition of P-gp by CsA increases the accumulation of NT in the brain. Administration of the antipsychotic drug risperidone (0.5 mg/kg s.c.), which is a P-gp substrate, instead of CsA did not exert any measurable influence on the blood-brain ratio of NT concentrations. In conclusion, the results show that drug-drug interaction at P-gp may influence the intracerebral NT concentration, but apparently, a major inhibition of P-gp is necessary to attain a measurable effect.

The role of metabolites in bioequivalence.

The role of metabolites in bioequivalence studies has been a contentious issue for many years. Many papers have published recommendations for the use of metabolite data based on anecdotal evidence from the results of bioequivalence studies. Such anecdotal evidence has validity, but the arguments lack weight because the "correct" answers are always unknown. A more promising area of exploration is recommendations based on simulated bioequivalence studies for which the "correct" answers are known, given the assumptions. A review of the literature, however, reveals scant evidence of attempts to apply to real data the pharmacokinetic principles on which the recommendations from simulated studies relied. We therefore applied those principles (based on estimates of intrinsic clearance after oral administration of the parent drug) to four bioequivalence studies from our archives, in which the parent drug and at least one metabolite were monitored. In each case, the outcome is discussed in the context of the complexity of the metabolic processes that impact on the parent drug and the metabolite(s) during the first passage from the intestinal lumen to the systemic circulation. Our observation is that no simple generalization can be made such that each drug/metabolite combination must be examined individually. Our recommendation, however, is that in the interests of safety, bioequivalence decision-making should be based on the parent drug whenever possible.

Inhibition of cytochrome P4502D6 activity with paroxetine normalizes the ultrarapid metabolizer phenotype as measured by nortriptyline pharmacokinetics and the debrisoquin test.

BACKGROUND: The ultrarapid metabolizer phenotype of the cytochrome P4502D6 (CYP2D6) enzyme has been considered a relevant cause of nonresponse to antidepressant drug therapy. Prescribing high doses of antidepressants to such patients leads to high concentrations of potentially toxic metabolites and an increased risk for adverse reactions. Normalization of the metabolic status of ultrarapid metabolizers by inhibition of CYP2D6 activity could offer a clinically acceptable method to successfully treat such patients with antidepressants. METHODS: Five ultrarapid metabolizers with a CYP2D6 gene duplication or triplication were treated with 25 mg nortriptyline twice a day for 3 consecutive weeks, alone during the first week and concomitantly with the CYP2D6 inhibitor paroxetine 10 mg or 20 mg twice a day, respectively, during the second and third weeks. After the third week, nortriptyline was discontinued and the subjects were treated with paroxetine 20 mg twice a day during the fourth study week. At the end of each study week, the steady-state pharmacokinetic parameters of nortriptyline or paroxetine were determined within the dose interval. In addition, the CYP2D6 phenotype was determined by debrisoquin (INN, debrisoquine) test at baseline and at the end of each study phase. Treatment-related adverse events were recorded during drug administration and for 1 week thereafter. RESULTS: All 5 subjects had very low (subtherapeutic) nortriptyline concentrations after 7 days' treatment with nortriptyline only. Addition of paroxetine 10 mg twice a day to the nortriptyline regimen resulted in a change in all individuals to the "normal" extensive debrisoquine metabolizer phenotype, and therapeutic plasma nortriptyline concentrations were achieved in 4 of 5 subjects after a 3 times mean increase in nortriptyline trough concentration (P =.0011). Doubling the paroxetine dose caused a 15 times mean increase in paroxetine trough concentration (P <.001), indicating strong inhibition by paroxetine of its own metabolism. The high paroxetine concentrations in 2 subjects caused them to have the poor debrisoquine metabolizer phenotype and resulted in a further increase in plasma nortriptyline trough concentration (P =.0099). A strong correlation (rank correlation coefficient [r(s)] = 0.89; P <.0001) was observed between paroxetine and nortriptyline trough concentrations. Paroxetine also significantly decreased the fluctuation of nortriptyline concentrations within the dose interval. One subject discontinued the study after the second study week because of adverse effects; otherwise, the study drugs were well tolerated. CONCLUSIONS: Paroxetine, with a daily dosage from 20 to 40 mg, is an effective tool in normalizing the metabolic status of CYP2D6 ultrarapid metabolizers.

Steady-state plasma levels of nortriptyline and its hydroxylated metabolites in Japanese patients: impact of CYP2D6 genotype on the hydroxylation of nortriptyline.

The authors investigated the impact of the CYP2D6 genotype on steady-state concentrations of nortriptyline (NT) and its metabolites, trans-10-hydroxynortriptyline (EHNT) and cis-10-hydroxynortriptyline in a Japanese population of psychiatric patients. Forty-one patients (20 men and 21 women) were orally administered nortriptyline hydrochloride. The allele frequencies of the CYP2D6*5 and CYP2D6*10 were 4.9% and 34.1%, respectively. Significant differences in NT concentrations corrected for dose and weight were observed between the subjects with no mutated alleles and those with one mutated allele (mean +/- SD for no mutated alleles vs. one mutated allele: 70.3 +/- 25.4 vs. 98.4 +/- 36.6 ng/mL x mg(-1) x kg(-1); t = 2.54, dcf = 33, p < 0.05) and between the subjects with no mutated alleles and two mutated alleles (no mutated alleles vs. two mutated alleles: 70.3 +/- 25.4 vs. 147 +/- 31.1 ng/mL x mg(-1) x kg(-1); t = 5.87, df = 19, p < 0.0001). Also, a significant difference in the NT/EHNT ratio, which is representative of the hydroxylation ratio of NT, was observed between the subjects with no mutated alleles and those with two mutated alleles (no mutated alleles vs. two mutated alleles: 0.82 +/- 0.30 vs. 2.71 +/- 0.84; t = 7.86, df = 19, p < 0.0001). Multiple regression analysis showed that the number of mutated alleles of CYP2D6, which was the only significant factor, accounted for 41% and 48% of the variability in log(NT corrected for dose and weight) and log(NT/EHNT), respectively.

Pharmacokinetics of nortriptyline and its 10-hydroxy metabolite in Chinese subjects of different CYP2D6 genotypes.

OBJECTIVES: To study the impact of the CYP2D6*10 allele on the disposition of nortriptyline in Chinese subjects. METHODS: A single dose of 25 mg nortriptyline was given orally to 15 healthy Chinese volunteers who were classified as extensive metabolizers after phenotyping with debrisoquin (INN, debrisoquine) and who were genotyped by allele-specific polymerase chain reaction. Five subjects were homozygous for CYP2D6*1, 5 subjects were homozygous for CYP2D6*10, and 5 subjects were heterozygous for these 2 alleles. Plasma concentrations of nortriptyline and its main metabolite 10-hydroxynortriptyline were measured by liquid chromatography-mass spectrometry, and the pharmacokinetics were studied during 168 hours after the dose. RESULTS: Subjects who were homozygous for CYP2D6*10 had significantly higher total areas under the plasma concentration-time curve (AUC), lower apparent oral clearances, and longer mean plasma half-life of nortriptyline than subjects in the CYP2D6*1/*1 and the heterozygous groups. For 10-hydroxynortriptyline, the AUC was lower and the plasma half-life was longer in subjects who were homozygous for CYP2D6*10 than in subjects in the other 2 groups. CONCLUSION: The CYP2D6*10 allele in Chinese subjects was associated with significantly higher plasma levels of nortriptyline compared with the CYP2D6*1 allele because of an impaired metabolism of nortriptyline to 10-hydroxynortriptyline, particularly in the subjects with the CYP2D6*10/*10 genotype. The results suggest that genotyping of CYP2D6 may be a useful tool in predicting the pharmacokinetics of nortriptyline.

High-performance liquid chromatography-mass spectrometry for the quantification of nortriptyline and 10-hydroxynortriptyline in plasma.

A highly sensitive and selective method for the quantification of nortriptyline and its major 10-hydroxy metabolite in plasma is described. The method is based on liquid-liquid extraction in combination with acid dehydration of the 10-hydroxy metabolite to the less polar 10,11-dehydronortriptyline. Deuterium labelled internal standards ([2H4]NT and [2H3]10-OH-NT) were used and the compounds were separated by reversed-phase HPLC and detected using atmospheric pressure chemical ionisation and mass spectrometry. The limit of quantification was 0.8 ng/ml for both compounds. A 1-ml volume of plasma was used for analysis in the concentration range 0.8-32 ng/ml. The within- and between-day coefficients of variation were 11% in the low, 1.6 ng/ml range, and 7% at 8 ng ml/ml. Using this method it was possible to quantify plasma concentrations for 168 h following a single oral dose of 25 mg of nortriptyline with good accuracy and precision.

10-Hydroxylation of nortriptyline in white persons with 0, 1, 2, 3, and 13 functional CYP2D6 genes.

OBJECTIVE: To investigate the disposition and effects of nortriptyline and its major metabolite 10-hydroxy-nortriptyline line in panels of white subjects with different CYP2D6 genotypes, including those with duplicated and multiduplicated CYP2D6*2 genes and to evaluate the contribution of the number of functional C gamma P2D6 alleles to the metabolism of nortriptyline, used here as a model drug for CYP2D6 substrates. METHODS: Oral single doses of 25 to 50 mg nortriptyline were given to five poor metabolizers of debrisoquin (INN; debrisoquine) with no functional CYP2D6 gene, five extensive metabolizers with one functional CY2D6 gene, five extensive metabolizers with two functional CYP2D6 genes, five ultrarapid metabolizers with duplicated CYP2D6*2 genes, and one ultrarapid metabolizer with 13 copies of the CYP2D6*2 gene. Plasma kinetics of nortriptyline and 10-hydroxynortriptyline were analyzed. Anticholinergic effects (inhibition of salivation and accommodation disturbances), sedation, blood pressure, and effect on supine and erect pulse rate were measured. RESULTS: There was a clear relation between the C gamma P2D6 genotype and the plasma kinetics of nortriptyline and 10-hydroxynortriptyline. The proportion between the apparent oral clearances of nortriptyline in the groups with 0, 1, 2, 3, and 13 functional genes was 1:1:4:5:17. The proportions between AUC(nortriptyline) to AUC(10-hydroxynortriptyline) ratios in the groups with 0, 1, 2, 3, and 13 functional genes were 36:25:10:4:1. Oral plasma clearance of nortriptyline and AUC(nortriptyline) to AUC(10-hydroxynortriptyline) ratio both correlated significantly with the debrisoquin metabolic ratio (rS = -0.89, p = 0.0001; rS = 0.92, p = 0.0001). Although ultrarapid metabolizer subjects were given double the nortriptyline dose (50 mg), inhibition of salivation was not more pronounced compared with the other genotype groups given 25 mg nortriptyline. CONCLUSION: The results of this study show the quantitative importance of the CYP2D6 genotype, especially the presence of multiple functional CYP2D6 genes for the pharmacokinetics of nortriptyline and 10-hydroxynortriptyline. Genotyping of subjects with multiple copies of functional genes may be of great value for differentiating ultrarapid metabolizers from patients who do not comply with the prescription and for assuring adequate drug choice and dosage for these patients.

Metabolism of amitriptyline with CYP2D6 expressed in a human cell line.

1. Expressed human cytochrome P450 enzyme CPY2D6 was used to metabolize amitriptyline (AMI). It was established that CYP2D6 not only catalyzed ring 10-hydroxylation of AMI, but also mediated its N-demethylation to nortriptyline (NT), as well as the formation of 10-hydroxy-NT from NT. When the metabolism of AMI by CYP2D6 was repeated in the presence of quinidine, none of the metabolites, 10-hydroxy-AMI, NT and 10-hydroxy-NT, was formed. 2. Biochemical parameters of NT formation from AMI were determined, yielding Km = 47.48 +/- 1.32 microM; Vmax = 3.95 +/- 0.11 nmol/h/mg protein. The same parameters were calculated for the formation of 10-hydroxy-AMI (E + Z-isomers) from AMI, yielding Km = 10.70 +/- 0.20 microM; Vmax = 8.99 +/- 0.47 nmol/h/mg protein. 3. The formation of 10-hydroxy-NT from AMI proceeded primarily via NT and to a much lesser extent via 10-hydroxy-AMI. 4. Quantitative analyses of AMI and its metabolites were difficult to reproduce when the metabolites were analysed underivatized. Two derivatization procedures, acetylation and trifluoroacetylation, were employed to improve assay reproducibility.

Pharmacokinetics of amitriptyline and its demethylated and hydroxylated metabolites in streptozocin-induced diabetic rats.

Plasma and brain levels of amitriptyline (AMI), its demethylated and hydroxylated metabolites were determined after acute IP administration of AMI (20 mg/kg) in streptozocin-induced diabetic Sprague-Dawley rats. Results showed 1. in plasma: rapid AMI absorption, but slow elimination; the proportion of AMI similar to those of the rest of compounds; the proportion of its demethylated metabolite, nortriptyline, 1.8-fold higher than that of 10-hydroxy-nortriptyline. 2. in brain: the proportions of AMI and nortriptyline were 9.5- and 2.6-fold higher respectively, than those of whole hydroxylated metabolites, which represented 7.4% of the total amount.

Plasma and brain pharmacokinetics of amitriptyline and its demethylated and hydroxylated metabolites after one and six half-life repeated administrations to rats.

The purposes of the present study were as follows: 1. After an acute intraperitoneal (IP) administration of amitriptyline (AMI) to male Sprague-Dawley rats we found that: (i) its absorption rate is rapid; (ii) its elimination half-life is much shorter than in humans; and (iii) its levels largely exceeded those of its metabolites. The most important metabolites being 10-hydroxynortriptyline and nortriptyline in plasma and brain, respectively. 2. After six (every half-life) repeated IP administrations: (i) AMI kinetic parameters were unchanged; and (ii) amounts of metabolites were significantly increased and the levels of AMI were lowered both in plasma and brain.